The expression of virulence determinants is tightly regulated so that they are produced at the right time and place during infection. Bacterial pathogens typically rely on two-component systems to modulate gene expression in response to environmental cues. The PhoP/PhoQ two-component system is the master regulator of Salmonella pathogenicity by governing the production of virulence proteins that are required in different tissues and cellular compartments. The PhoQ protein is a sensor for extracytoplasmic Mg2+ that controls the phosphorylated state of the PhoP protein, a transcription factor whose has higher affinity for its target DNAs when phosphorylated. We have determined that the activation of the PhoP/PhoQ system is transient despite the continuous presence of Inducing signals, and that the PhoP protein can regulate transcription from a wide variety of promoters. This proposal describes experiments aimed at understanding how the PhoP/PhoQ system is controlled, how the PhoP protein regulates transcription from different classes of promoters and in conjunction with other regulatory proteins, and to determine the virulence significance of PhoP-mediated expression of other regulatory proteins, such as SlyA and SsrB, that are required for Salmonella's ability to survive in macrophages. An accomplishment of these goals will take us closer to understanding the mechanisms by which a microbe can modify its gene expression repertoire in response to environmental cues. Moreover, they will help the study of other pathogenic organisms and other two-component systems because the PhoP/PhoQ system is necessary for virulence in Yersinia spp. and Shigella flexneri, and because the biochemical activities of two-component systems are largely conserved. [unreadable] [unreadable] [unreadable]